Vaccination is an extremely complex process, which mimics molecular and cellular processes occurring during induction of natural immune responses after injury and microbiological infections and which involves two strictly local reactions: one at the level of the inoculation site and one on the level of the draining lymph node(s).
In such natural immune responses, interactions between cells of the immune system are regulated and directed by the exchange of cytokines in the cytokine network. Most of our knowledge about the interactions between the cells of the immune system and about the actions of cytokines in these interactions has been elaborated by in vitro experimentation. Based on said results it is generally accepted that many types of cells of the innate and the adaptive immune system are “professional” secretors.
They have the intracellular machinery to produce, store and release a variety of cytokines, chemokines and other secreted substances (mediators, e.g. Serotonin, Histamine).
Results of recent research (Huse, Morgan, B. F. Lillemeier. M. S. Kuhns, D. S. Chen & M. M. Davis) show that T cells use two directionally distinct pathways for cytokine secretion. 2006 Nature Immunology 7, 247-255; Stanley, C. Amanda and Lacy Paige Pathways for Cytokine secretion. 2010. Reviews. Physiology 25, 218-229) have shown that the cytokine molecules can be released                either by exocytosis to the outside of the cells or directed to synapses through which cells are in contact with each other, e.g. to the immunological synapses between T lymphocytes and antigen presenting cells,        or through multi-directional pathways (e.g. by constitutive cytokine release by carrier vesicles that transport cargo to the plasma membrane for immediate (within minutes of stimulation) release, or by piecemeal degranulation of small secretory vesicles), or by directed bimodal secretion (the release of different cargo (=cytokines) in different directions) enables cells to engage simultaneously in both “public” (to all the cells in proximity) and “private” (to a cell in contact) intercellular “conversation”.        
Consequently, cytokine concentrations at the site of injury or inflammation will be dependent;                on the momentary position of the cells in relation to each other;        on the activation/stimulation state of the cells involved;        on the way the cytokine molecules are released (timing, trickle or burst); and        on the mobility of released cytokine molecules in the extra-cellular fluid (speed of diffusion, concentration gradient, distance the molecules can migrate).        
Present pharmacology has not yet found ways and procedures to interact and mimic these inter-cellular processes of the immune system at the local cellular level. So far practically all pharmaceutical interactions are based on systemic (i.v., s.c., i.d.) applications. However, if molecular messenger molecules, such as cytokines, are applied systemically, they tend to flood the organism and actually render local cell-to-cell interactions impossible. Nevertheless, in specific circumstances, they can induce and modify immune reactions, e.g. in the treatment of cancer by activation of lymphocytes that have infiltrated tumor lesions or metastases (TIL: Tumor-Infiltrating Lymphocytes) and have been rendered inert by the tumor cells.
Cells of the immune system are capable of producing, storing and releasing more than a single cytokine. It can further be assumed that each cytokine is released for a different purpose. Thus, it is rather unlikely that all the cytokines are stored in the same vesicles and always released together. This has also been shown experimentally. The mechanisms of this finely tuned molecular machinery that regulates secretion of dozens of cytokines, chemokines and other small molecule mediators (e.g. by mast cells) is not understood today but must be responsible for the release processes.
Cytokine molecules are released when a cytokine-containing vesicle fuses with the cell's membrane and opens to the outside or through a synapse into another cell. Such a process cannot result in a slow continuous flow of cytokine molecules from the cell to its outside, but has to be burst-like, “shooting” the cytokine molecules into the extra-cellular environment or through a synapse into a neighboring cell.
In the vesicles, the cytokine molecules are densely packed. Consequently near the point of release, close to the surface of the cells, cytokine concentrations are extremely high. But after a few cell diameters of diffusion they will be reduced to concentrations required for binding to cytokine receptors. Some of the cytokine molecules might reach the vascular system; most will just be lost in the extra-cellular space.
In 1996 David R. Kaplan published a review (Kaplan, David R. Autocrine secretion and the physiological concentration of cytokines. 1996 Trends. Immunology Today 17, 303-304), in which he has summarized data from other researchers. Based on these data he has made the following estimate:                1. a single activated T lymphocytes is capable of releasing about 0.04 pg of IL-2 per hour, corresponding to about 106 IL-2 molecules.        2. these 0.04 pg of IL-2 are stored in 20-2,000 vesicles in extremely high density of 1-100 mM (corresponding to 12-1.200 gram of IL-2 per litre).        3. after fusion of a cytokine-containing vesicle with the cell's membrane and opening of the vesicle to the outside of the cells, IL-2 concentration will be in the same range of 1-100 mM.        4. This concentration is much too high for binding to the T cell receptor on the same cell's membrane.        5. after diffusion during about 100 seconds and in a few cell diameters distance from the secreting lymphocyte, IL-2 concentration reaches the level required for binding to cytokine receptors.        
Consequently, in an immune reaction, each activated T cell would burst-out a shower of about 1,000,000 IL-2 molecules released from between 20 and 2,000 vesicles of different sizes (=between 500-50,000 Molecules per vesicle)
In order to imitate such a reactions, it is not sufficient to release a trickle of IL-2 molecules, as is the case with cytokine gene-transfected tumor cells, as they have been applied in cancer vaccines (e.g. Nemunaitis et al. J. Natl. Cancer Inst. (2004) 96:326-331).
Also the local injection of several hundred micrograms of cytokines as is done in systemic cytokine treatment is far away from the natural process: a huge shower of trillions (more than 1,000,000,000,000) of cytokine molecules is not capable of imitating the natural release pattern of a professional secretory cell.
The local release of such a huge amount of cytokine molecules, corresponding to the simultaneous release by several millions of activated lymphocytes, will never happen under natural conditions and will cause absolute chaos at and around the inoculation site and—after reaching the vascular system—might also cause havoc in distant locations.
Direct application of IL-2 under the trade name Proleukin (Chiron Corp.) has been approved by the United States FDA for the treatment of adults with metastatic renal cell carcinoma and metastatic melanoma. Already from the early stages of research into IL-2 containing pharmaceutical compositions, it was apparent that aggregation-preventing agents are needed to ensure solubility of IL-2. For example, in U.S. Pat. No. 4,604,377, which describes the earliest pharmaceutical compositions of IL-2, indicates that about 100 to about 250 μg sodium dodecyl sulfate (SDS) should be present to avoid IL-2 aggregation and ensure solubility.
EP1688146, which describes amongst others the process to obtain the Proleukin composition, further details the importance of the amount of SDS in the composition. The needed amount of SDS is considered to be 95 to 250 μg per mg of IL-2, at which concentration the IL-2 is present in microaggregates of approximately 25-60 IL-2 molecules per aggregate. The preferred amount of SDS is, as also present in the Proleukin formulation, 160 μg SDS per mg IL-2, which leads to microaggregates of about 27 molecules IL-2, with a diameter of about 12 nm. As the SDS concentration drops below 95 μg/mg, the sizes of the aggregates rise sharply, leading to worse in vivo pharmacokinetics. The clearance rate in rats was even found to be 30-fold higher for a composition containing 25 μg SDS per mg IL-2 compared to the preferred composition of 160 μg SDS per mg IL-2.
An interesting variation of direct injection of IL-2 such as injection of Proleukin, is presented in U.S. Pat. No. 6,406,689. In that patent, the aforementioned Proleukin IL-2 formulation (comprising SDS in a range of 95 to 250 μg per mg) is adsorbed to aluminum hydroxide. Thereafter, it is mixed with irradiated tumor cells and injected into mice wherein renal carcinoma was induced. While survival rates where higher for IL-2 in combination with irradiated tumor cells compared to irradiated tumor cells alone, survival rates further increased when IL-2 was adsorbed to aluminum hydroxide.
The inventor of the present invention has surprisingly found that generating cytokine macro-aggregates of e.g. IL-2 and adsorbing these to a depot material, such as aluminum hydroxide, leads to compositions with improved pharmaceutical properties. Instead of the expected worse in vivo pharmacokinetics associated with these systemically applied macro-aggregates as described in EP1688146, these macro-aggregates actually improve in vivo outcome compared to micro-aggregates, when adsorbed to a depot material and applied as adjuvants in vaccines, as will be described hereinafter.
It has thus been an object of the present invention to provide an improved composition for inducing immune responses, in particular for treating tumors, such as for vaccination against cancer.
Further to the surprise of the inventor, such a composition comprising cytokine macroaggregates adsorbed to a depot material and antigenic material appears to closely mimic the natural immune system. As will be described in more detail hereinafter, it has been found that cytokine molecules or small aggregates are released from the macro-aggregates in localized bursts, similar to the vesicle release observed in the secretory events of cells of the immune system. Furthermore, said vaccine formulation is being characterized in participating in immune-stimulatory processes not only locally at the inoculation site, but also in the lymph node(s) draining the area of the inoculation site. Without wishing to be bound by theory, it is currently assumed that the CD25A receptor might play a role in this effect. Mature CD86+ dendritic cells bear the low affinity CD25A IL-2 receptor. Due to its low affinity properties the CD25A low affinity receptor on the CD86+ dendritic cells binds IL-2 aggregates rather than isolated IL-2 molecules. This might be the reason that the CD86+ dendritic cells carry both, antigenic fragments and IL-2 aggregates, to the lymph nodes.
Thus, it is also an object of the present invention to provide the use of a composition comprising such cytokine macroaggregates in mimicking the natural immune system and stimulating it, not only at the inoculation site, but also at the lymph node(s) draining the area of the inoculation site.